Ventricular arrhythmias often cause sudden cardiac death, a leading cause of death in the United States. Although the detailed mechanisms are not completely understood, there is growing evidence that abnormal calcium cycling plays a fundamental role in the pathogenesis of fatal arrhythmias. Therefore, understanding how calcium handling is regulated and disrupted in the diseased hearts is critical for developing effective antiarrhythmic therapies. In addition to defective calcium homeostasis, increased mitochondrial-derived reactive oxygen species (mdROS) production is another common feature of arrhythmia-prone hearts. We hypothesize that mdROS may be a key factor involved in the abnormal calcium transients and electrical activities in cardiac cells with mitochondrial dysfunction and this redox signaling is mediated by mitochondria-sarcoplasmic reticulum (SR) tethering. The proposed hypothesis is inspired by previous studies showing that: 1) intracellular Ca2+ cycling is largely determined by the redox sensitive Ca2+ channels located on SR, which are in close proximity to mitochondria; 2) mitochondrial Ca2+ uptake relies on mitochondria-SR tethering orchestrated by mitofusin 2 (Mfn2); and 3) suppressing mitochondria Ca2+ uptake not only impairs mitochondrial energetics but also exacerbates cytosolic Ca2+ dysregulation. The hypothesis will be tested by systematically examining the mdROS-induced Ca2+ dysregulation and the role of mitochondria-SR tethering in mediating this redox modulation in the control and diseased (e.g. Mfn2 knockout and heart failure) hearts. Specifically, we will: 1) examine the molecular mechanisms underlying mdROS-induced SR Ca2+ release using an innovative cardiac specific Mfn2 KO mouse model and super- resolution imaging techniques; 2) examine the influence of mdROS on mitochondria-SR tethering and Ca2+ cycling in pressure-overloaded hearts and test if MitoQ, a mitochondrial-targeted ROS scavenger, could reduce the risk of fatal cardiac arrhythmias in the failing hearts by suppressing mdROS production, preventing mitochondrial dysfunction, and alleviating abnormal Ca2+ regulation; and 3) develop a novel computational model of mitochondria-SR tethering to complement experimental studies to understand the role of interorganellar redox signaling in regulating mdROS-induced Ca2+ release in the cardiomyocytes under various pathological conditions. Successful completion of the proposed studies will provide critical insights into the mechanisms by which mitochondrial redox signaling modulates cytosolic Ca2+ cycling and electrical activities in the cardiomyocytes. It will also act as a foundation for future translational studies designed to target these mechanisms for the development of novel antiarrhythmic therapies.